The goal of this proposal is to test whether principles of protein interaction that have been inferred from natural systems can be used in protein engineering to design useful erythropoietin (EPO)-based molecules. Due to its stimulation of erythropoiesis and anti-inflammatory pathways, EPO is an attractive treatment option for anemia and autoimmune disease. However, its clinical use is inhibited by its off-target affects, including thrombosis and stimulation of tumor growth. To address the need for EPO-based drugs with greater specificity for distinct cell types, a synthetic biology approach can be found in how cells use multi-component receptor complexes to specifically target cytokines. For example, the interleukin 2 receptor is composed of three subunits, one of which acts as a high-affinity targeting domain for the other two to create an overall high-affinity receptor [50]. I hypothesize that this natural mechanism for precise protein targeting can be translated to the engineering of synthetic EPO-based therapeutics with improved targeting of erythroid progenitors or macrophages, for the treatment of anemia or autoimmune disease, respectively. I will build chimeric proteins composed of a mutated form of EPO with reduced affinity for its receptor, a targeting element that binds strongly to the target cell, and a linker that allows simultaneous binding. I hypothesize that by combining quantitative information about binding kinetics with structural information about protein ligands and cell surface receptors, the resulting proteins (chimeric activators) should activate signal transduction only in cells with receptors for both elements. The targeting element should mediate initial binding, and the mutated EPO should subsequently bind due to its high local concentration at the cell surface, despite its decreased binding affinity. This work will provide valuable information about the quantitative aspects of designing targeted protein-based drugs, the diverse biological roles of EPO and its value as a pleiotropic therapeutic, and may also enable new methods for engineering cell type-specific therapies. Overall, the findings will have implications for improving the quality of life and diseae prognosis in patients suffering from anemia or autoimmune disease.